DESIGN OF A MEMBRANE-PROTEIN FOR SITE-SPECIFIC PROTEOLYSIS - PROPERTIES OF ENGINEERED FACTOR XA PROTEASE SITES IN THE LACTOSE PERMEASE OF ESCHERICHIA-COLI
M. Sahintoth et al., DESIGN OF A MEMBRANE-PROTEIN FOR SITE-SPECIFIC PROTEOLYSIS - PROPERTIES OF ENGINEERED FACTOR XA PROTEASE SITES IN THE LACTOSE PERMEASE OF ESCHERICHIA-COLI, Biochemistry, 34(4), 1995, pp. 1107-1112
Lactose permease is a polytopic membrane transport protein with 12 hyd
rophobic transmembrane domains connected by hydrophilic loops on the c
ytoplasmic and periplasmic sides of the membrane. By the use of an act
ive permease mutant devoid of Cys residues (C-less permease), single r
ecognition sites (Ile-Glu-Gly-Arg) for the protease factor Xa (fXa) we
re engineered into hydrophilic loops 7, 8, and 10 in the C-terminal ha
lf of the protein. Mutants carrying single sites inserted at position
255, 259 (loop 7), 283, 286 (loop 8), or 341 (loop 10) exhibit signifi
cant lactose accumulation (30-70% of C-less permease) and normal level
s of expression in the membrane. However, despite solubilization in do
decyl beta-D-maltoside, none of the mutant permeases is proteolyzed by
fXa to a significant extent. Insertion of two recognition sites in ta
ndem at position 255 results in partial cleavage, and remarkably, intr
oduction of three sites in tandem leads to complete proteolysis by fXa
. Importantly, mutants with two or three fXa sites at position 255 acc
umulate lactose to high levels (70% of C-less) and are present in the
membrane in amounts comparable to that of C-less permease. The results
indicate that hydrophilic loops 7, 8, and 10 are buried in the tertia
ry structure of the permease where they are inaccessible to protease.
Insertion of tandem sites probably facilitates proteolysis by causing
loops to become more accessible to the aqueous phase and by increasing
the local concentration of protease recognition sites. The approach s
hould be applicable to other polytopic membrane proteins.